ACCURACY AND LIMITATIONS OF PULSED FIELD GEL-ELECTROPHORESIS IN SIZING PARTIAL DELETIONS OF THE FACTOR-VIII GENE

Abstract

Pulsed field gel electrophoresis (PFGE) has been recently used to separate DNA fragments ranging from 100 to 2000 kb in size. In order to assess the accuracy of the sizes of the DNA fragments and the resolving capability of this technique, we used PFGE combined with Southern blotting and probe hybridization techniques to determine the size and approximate location of four partial deletions of the factor VIII gene. This gene was chosen because of its large size (186 kb) and the availability of hemophiliac patients with well-characterized partial deletions. The sizes of three deletions estimated by PFGE (55 kb, 60 kb and 133 to 145 kb) were within 10% of the sizes calculated from conventional restriction analysis. Therefore, concatemers of lambda DNA and intact chromosomal DNA of Saccharomyces cerevisiae provide a relatively accurate system (within 10%) for sizing mammalian DNA fragments that are 100 to 550 kb in size. However, analysis of the fourth deletion (9 to 12 kb) revealed that it is difficult to detect changes in the size of mammalian DNA fragments of 8% or less using Southern blotting and PFGE.